Expression of biologically active polypeptides in duckweed

ABSTRACT

Methods, nucleic acid sequences, and transformed duckweed plant or duckweed nodule cultures for the expression and the secretion of biologically active polypeptides from genetically engineered duckweed are provided. Expression of recombinant polypeptides in duckweed is improved by modifying the nucleotide sequence of the expression cassette encoding the polypeptide for improved expression in duckweed. Recovery of biologically active polypeptides from duckweed is improved by linking the biologically active polypeptide to a signal peptide that directs the secretion of the polypeptide into the culture medium.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. application Ser. No.09/915,873, filed Jul. 26, 2001; which claims the benefit of U.S.Provisional Application Ser. No. 60/221,705, filed Jul. 31, 2000, andU.S. Provisional Application Ser. No. 60/293,330, filed May 23, 2001;each of which is hereby incorporated in its entirety by referenceherein.

FIELD OF THE INVENTION

The present invention relates to methods and compositions for theexpression and the secretion of biologically active polypeptides fromgenetically engineered duckweed.

BACKGROUND OF THE INVENTION

The duckweeds are the sole members of the monocotyledonous familyLemnaceae. The four genera and 34 species are all small, free-floating,fresh-water plants whose geographical range spans the entire globe(Landolt (1986) Biosystematic Investigation on the Family of Duckweeds:The Family of Lemnaceae—A Monograph Study Geobatanischen Institut ETH,Stiftung Rubel, Zurich). Although the most morphologically reducedplants known, most duckweed species have all the tissues and organs ofmuch larger plants, including roots, stems, flowers, seeds and fronds.Duckweed species have been studied extensively and a substantialliterature exists detailing their ecology, systematics, life-cycle,metabolism, disease and pest susceptibility, their reproductive biology,genetic structure, and cell biology. (Hillman (1961) Bot. Review 27:221; Landolt (1986) Biosystematic Investigation on the Family ofDuckweeds: The Family of Lemnaceae—A Monograph Study GeobatanischenInstitut ETH, Stiftung Rubel, Zurich).

The growth habit of the duckweeds is ideal for microbial culturingmethods. The plant rapidly proliferates through vegetative budding ofnew fronds, in a macroscopic manner analogous to asexual propagation inyeast. This proliferation occurs by vegetative budding from meristematiccells. The meristematic region is small and is found on the ventralsurface of the frond. Meristematic cells lie in two pockets, one on eachside of the frond midvein. The small midvein region is also the sitefrom which the root originates and the stem arises that connects eachfrond to its mother frond. The meristematic pocket is protected by atissue flap. Fronds bud alternately from these pockets. Doubling timesvary by species and are as short as 20-24 hours (Landolt (1957) Ber.Schweiz. Bot. Ges. 67: 271; Chang et al. (1977) Bull. Inst. Chem. Acad.Sin. 24:19; Datko and Mudd (1970) Plant Physiol. 65:16; Venkataraman etal. (1970) Z. Pflanzenphysiol. 62: 316).

Intensive culture of duckweed results in the highest rates of biomassaccumulation per unit time (Landolt and Kandeler (1987) The Family ofLemnaceae—A Monographic Study Vol. 2: Phytochemistry, Physiology,Application, Bibliography, Veroffentlichungen des GeobotanischenInstitutes ETH, Stiftung Rubel, Zurich), with dry weight accumulationranging from 6-15% of fresh weight (Tillberg et al. (1979) Physiol.Plant. 46:5; Landolt (1957) Ber. Schweiz. Bot. Ges. 67:271; Stomp,unpublished data). Protein content of a number of duckweed species grownunder varying conditions has been reported to range from 15-45% dryweight (Chang et al (1977) Bull. Inst. Chem. Acad. Sin. 24:19; Chang andChui (1978) Z. Pflanzenphysiol. 89:91; Porath et al. (1979) AquaticBotany 7:272; Appenroth et al. (1982) Biochem. Physiol. Pflanz.177:251). Using these values, the level of protein production per literof medium in duckweed is on the same order of magnitude as yeast geneexpression systems.

Sexual reproduction in duckweed is controlled by medium components andculturing conditions, including photoperiod and culture density. Flowerinduction is a routine laboratory procedure with some species. Plantsnormally self-pollinate, and selfing can be accomplished in thelaboratory by gently shaking cultures. By this method, inbred lines ofLemna gibba have been developed. Spontaneous mutations have beenidentified (Slovin and Cohen (1988) Plant Physiol. 86, 522), andchemical and gamma ray mutagenesis (using EMS or NMU) have been used toproduce mutants with defined characteristics. Outcrossing of L. gibba istedious but can be done by controlled, hand pollination. The genome sizeof the duckweeds varies from 0.25-1.63 pg DNA/2C with chromosome countsranging from 20 to 80 and averaging about 40 across the Lemnaceae(Landolt (1986) Biosystematic Investigation on the Family of Duckweeds:The family of Lemnaceae—A Monograph Study Geobatanischen Institut ETH,Stiftung Rubel, Zurich). Ploidy levels are estimated to range from 2-12C. Id. Genetic diversity within the Lemnaceae has been investigatedusing secondary products, isozymes, and DNA sequences (McClure andAlston (1966) Nature 4916:311; McClure and Alston (1966) Amer. J Bot.53:849; Vasseur et al. (1991) Pl. Syst. Evol. 177:139 (1991); Crawfordand Landolt (1993) Syst. Bot. 10:389).

Duckweed plant or duckweed nodule cultures can be efficientlytransformed with an expression cassette containing a nucleotide sequenceof interest by any one of a number of methods includingAgrobacterium-mediated gene transfer, ballistic bombardment, orelectroporation. Stable duckweed transformants can be isolated bytransforming the duckweed cells with both the nucleotide sequence ofinterest and a gene that confers resistance to a selection agent,followed by culturing the transformed cells in a medium containing theselection agent. See U.S. Pat. No. 6,040,498 to Stomp et al.

A duckweed gene expression system provides the pivotal technology thatwould be useful for a number of research and commercial applications.For plant molecular biology research as a whole, a differentiated plantsystem that can be manipulated with the laboratory convenience of yeastprovides a very fast system in which to analyze the developmental andphysiological roles of isolated genes. For commercial production ofvaluable polypeptides, a duckweed-based system has a number ofadvantages over existing microbial or cell culture systems. Plantsdemonstrate post-translational processing that is similar to mammaliancells, overcoming one major problem associated with the microbial cellproduction of biologically active mammalian polypeptides, and it hasbeen shown by others (Hiatt (1990) Nature 334:469) that plant systemshave the ability to assemble multi-subunit proteins, an ability oftenlacking in microbial systems. Scale-up of duckweed biomass to levelsnecessary for commercial production of recombinant proteins is fasterand more cost efficient than similar scale-up of mammalian cells, andunlike other suggested plant production systems, e.g., soybeans andtobacco, duckweed can be grown in fully contained and controlled biomassproduction vessels, making the system's integration into existingprotein production industrial infrastructure far easier.

These characteristics make duckweed an ideal choice to develop as anefficient, plant-based system for the production of recombinantproteins. Accordingly, the present invention provides methods andcompositions that increase the efficiency of the duckweed geneexpression system as a tool for producing biologically activepolypeptides.

SUMMARY OF THE INVENTION

The present invention is drawn to methods and compositions for theexpression and recovery of biologically active recombinant polypeptides,using duckweed as the expression system. One aspect of the presentinvention provides a method for enhanced expression levels ofbiologically active polypeptides in duckweed, resulting in an increasedpolypeptide yield and enabling the production of useful quantities ofvaluable biologically active polypeptides in this system. Another aspectof the invention discloses methods for the directed secretion ofbiologically active polypeptides from genetically engineered duckweedplant or duckweed nodule cultures. Secretion of the expressedpolypeptide facilitates its recovery and prevents the loss of activitythat might result from the mechanical grinding or enzymatic lysing ofthe duckweed tissue.

In one embodiment, the invention encompasses a method of producing abiologically active recombinant polypeptide in a duckweed plant cultureor a duckweed nodule culture, comprising the steps of: (a) culturingwithin a duckweed culture medium a duckweed plant culture or a duckweednodule culture, wherein said duckweed plant culture or said duckweednodule culture is stably transformed to express said biologically activerecombinant polypeptide, and wherein said biologically activerecombinant polypeptide is expressed from a nucleotide sequencecomprising a coding sequence for the polypeptide and an operably linkedcoding sequence for a signal peptide that directs secretion of thepolypeptide into the culture medium; and (b) collecting saidbiologically active polypeptide from the duckweed culture medium. Insome embodiments of this method, the nucleotide sequence has at leastone attribute selected from the group consisting of: (a)duckweed-preferred codons in the coding sequence for said polypeptide;(b) duckweed-preferred codons in the coding sequence for said signalpeptide; (c) a translation initiation codon that is flanked by aplant-preferred translation initiation context nucleotide sequence; and(d) an operably linked nucleotide sequence comprising a plant intronthat is inserted upstream of the coding sequence. In some embodiments ofthis method, the biologically active recombinant polypeptide is secretedinto the duckweed culture medium.

In another embodiment, the invention encompasses a method of secreting abiologically active recombinant polypeptide in a duckweed plant cultureor a duckweed nodule culture, comprising the steps of: (a) culturingwithin a duckweed culture medium a duckweed plant culture or a duckweednodule culture, wherein said duckweed plant culture or said duckweednodule culture is stably transformed to express said biologically activerecombinant polypeptide, and wherein said biologically activerecombinant polypeptide is expressed from a nucleotide sequencecomprising a coding sequence for the polypeptide and an operably linkedcoding sequence for a signal peptide that directs secretion of thepolypeptide into the culture medium; and (b) collecting saidbiologically active polypeptide from the duckweed culture medium. Insome embodiments of this method, the nucleotide sequence has at leastone attribute selected from the group consisting of: (a)duckweed-preferred codons in the coding sequence for said polypeptide;(b) duckweed-preferred codons in the coding sequence for said signalpeptide; (c) a translation initiation codon that is flanked by aplant-preferred translation initiation context nucleotide sequence; (d)an operably linked nucleotide sequence comprising a plant intron that isinserted upstream of the coding sequence; and (e) an operably linkednucleotide sequence comprising the ribulose-bis-phosphate carboxylasesmall subunit 5B gene of Lemna gibba. In some embodiments of thismethod, the biologically active recombinant polypeptide is secreted intothe duckweed culture medium.

In some embodiments of the above methods, the signal peptide is selectedfrom the group consisting of: (a) the human α-2b-interferon signalsequence; (b) the Arabidopsis thaliana chitinase signal sequence; (c)the rice α-amylase signal sequence; (d) the modified rice α-amylasesequence; (e) a duckweed signal sequence; (f) the human growth hormonesignal sequence; and (g) a signal sequence native to the biologicallyactive recombinant polypeptide. In one embodiment of the method, thesignal peptide is the rice α-amylase signal peptide whose sequence isset forth in SEQ ID NO:3.

In some embodiments of the above methods, the duckweed-preferred codonsare Lemna-preferred codons. In further embodiments, theduckweed-preferred codons are Lemna gibba-preferred codons or Lemnaminor-preferred codons. In further embodiments, at least one codingsequence selected from the coding sequence for the biologically activerecombinant polypeptide and the coding sequence for the signal peptidecomprises between 70-100% Lemna gibba-preferred codons or Lemnaminor-preferred codons.

In other embodiments, the invention encompasses a method of producing abiologically active recombinant polypeptide, comprising the steps of:(a) culturing a duckweed plant culture or a duckweed nodule culture,wherein said duckweed plant culture or said duckweed nodule culture isstably transformed to express said biologically active recombinantpolypeptide, and wherein said biologically active recombinantpolypeptide is encoded by a nucleotide sequence that has been modifiedfor enhanced expression in duckweed, and (b) collecting saidbiologically active polypeptide from said duckweed plant culture or saidduckweed nodule culture. In some embodiments of this method, thenucleotide sequence that has been modified for enhanced expression induckweed has at least one attribute selected from the group consistingof: (a) duckweed-preferred codons in the coding sequence for saidbiologically active recombinant polypeptide; (b) a translationinitiation codon that is flanked by a plant-preferred translationinitiation context nucleotide sequence; and (c) an operably linkednucleotide sequence comprising a plant intron that is inserted upstreamof the coding sequence.

In some embodiments of this method, the duckweed-preferred codons areLemna-preferred codons. In further embodiments, the duckweed-preferredcodons are Lemna gibba-preferred codons or Lemna minor-preferred codons.In further embodiments, the coding sequence comprises between 70-100%Lemna gibba-preferred codons or Lemna minor-preferred codons.

In some embodiments of the above methods, the plant-preferredtranslation initiation context nucleotide sequence consists of thenucleotide sequence “ACC” or “ACA”, wherein said context is positionedimmediately adjacent to the 5′ end of the translation initiation codon.

In some embodiments of the above methods, the operably linked nucleotidesequence comprising said plant intron is the sequence set forth in SEQID NO: 1.

In some embodiments of any of the above methods, the duckweed frondculture or duckweed nodule culture expresses and assembles all of thesubunits of a biologically active multimeric protein. In furtherembodiments, the multimeric protein is selected from the groupconsisting of collagen, hemoglobin, P450 oxidase, a monoclonal antibody,or a Fab′ antibody fragment.

In some embodiments of any of the above methods, the biologically activerecombinant polypeptide is a mammalian polypeptide. In furtherembodiments, the mammalian polypeptide is a therapeutic polypeptide. Insome embodiments, the mammalian polypeptide is selected from the groupconsisting of: insulin, growth hormone, α-interferon, β-interferon, ,β-glucocerebrosidase, β-glucoronidase, retinoblastoma protein, p53protein, angiostatin, leptin, monoclonal antibodies, cytokines,receptors, human vaccines, animal vaccines, plant polypeptides, andserum albumin.

In some embodiments of any of the above methods, the biologically activerecombinant polypeptide is α-2b-interferon. In further embodiments, theα-2b-interferon is human α-2b-interferon. In further embodiments, thehuman α-2b-interferon has the amino acid sequence set forth in SEQ IDNO:4 or SEQ ID NO:5.

In some embodiments of any of the above methods, the biologically activerecombinant polypeptide is a biologically active variant ofα-2b-interferon, wherein said biologically active variant has at least80% sequence identity with SEQ ID NO:4 or SEQ ID NO:5.

In some embodiments of any of the above methods, the biologically activerecombinant polypeptide is an enzyme.

In other embodiments, the invention encompasses the stably transformedduckweed plant culture or duckweed nodule culture of any of the abovemethods. In further embodiments, the stably transformed duckweed plantculture or duckweed nodule culture is selected from the group consistingof the genus Spirodela, genus Wolffia, genus Wolfiella, and genus Lemna.In further embodiments, the stably transformed duckweed plant culture orduckweed nodule culture is selected from the group consisting of Lemnaminor, Lemna miniscula, Lemna aequinoctialis, and Lemna gibba.

In other embodiments, the invention encompasses a nucleic acid moleculecomprising a nucleotide sequence encoding an amino acid sequenceselected from the group consisting of: (a) the amino acid sequence setforth in SEQ ID NO:4; (b) the amino acid sequence set forth in SEQ IDNO:5; (c) the amino acid sequence of a biologically active variant ofthe amino acid sequence shown in SEQ ID NO:4, wherein said biologicallyactive variant has at least about 80% sequence identity with the aminoacid sequence set forth in SEQ ID NO:4; and (d) the amino acid sequenceof a biologically active variant of the amino acid sequence shown in SEQID NO:5, wherein said biologically active variant has at least about 80%sequence identity with the amino acid sequence set forth in SEQ ID NO:5;wherein the nucleotide sequence comprises duckweed-preferred codons. Infurther embodiments, the nucleotide sequence is the nucleotide sequenceset forth in SEQ ID NO:2.

In other embodiments, the invention encompasses a nucleic acid moleculecomprising a nucleotide sequence encoding a signal peptide selected fromthe group consisting of: (a) the rice α-amylase signal peptide aminoacid sequence set forth in SEQ ID NO:6; and (b) the modifiedrice-amylase signal peptide amino acid sequence set forth in SEQ IDNO:7; wherein the nucleotide sequence comprises duckweed-preferredcodons. In further embodiments, the nucleotide sequence is thenucleotide sequence set forth in SEQ ID NO: 3.

In other embodiments, the invention encompasses a nucleic acid moleculefor the expression and secretion of human α-2b-interferon in duckweedcomprising the signal peptide-encoding nucleotide sequence given in SEQID NO:3, and the mature human α-2b-interferon-encoding nucleotidesequence given in SEQ ID NO:5, wherein the signal peptide-encodingnucleotide sequence and said mature human α-2b-interferon-encodingnucleotide sequence are operably linked. In further embodiments, thenucleic acid molecule additionally comprises the intron-comprisingnucleotide sequence given in SEQ ID NO: 1, wherein saidintron-comprising nucleotide sequence is operably linked to said signalpeptide-encoding nucleotide sequence and said mature humanα-2b-interferon-encoding nucleotide sequence.

In one embodiment, the present invention provides a method of enhancingthe expression of a biologically active polypeptide in duckweed. Themethod comprises culturing a duckweed plant culture or a duckweed noduleculture, wherein said duckweed plant culture or said duckweed noduleculture is stably transformed to express said biologically activepolypeptide and wherein said biologically active polypeptide isexpressed from a nucleotide sequence comprising a coding sequence forthe biologically active polypeptide and an operably linked nucleotidesequence comprising the leader from the ribulose-bis-phosphatecarboxylase small subunit gene of Lemna gibba.

These and other aspects of the present invention are disclosed in moredetail in the description of the invention given below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1

FIG. 1 show the interferon levels (as determined by a solid phasesandwich immunoassay) in the media and tissue of a transformed duckweedculture, as described Example 1.

FIG. 2

FIG. 2 show the interferon levels (as determined by a solid phasesandwich immunoassay) in the media and tissue of a transformed duckweedcultures, as described in Example 2.

DETAILED DESCRIPTION OF THE INVENTION

In the present invention, methods are disclosed for improving theexpression and the recovery of biologically active polypeptides fromgenetically engineered duckweed plants and duckweed nodule cultures.These methods comprise the steps of

(1) culturing a stably transformed duckweed plant or a duckweed noduleculture that expresses at least one biologically active polypeptide,wherein the polypeptide is encoded by a nucleotide sequence that hasbeen modified for enhanced expression in duckweed, and recovering thebiologically active polypeptide from the duckweed culture. Modificationsto the nucleotide sequence that are encompassed by this inventioninclude, but are not limited to, the use of duckweed-preferred codonsfor the coding sequence, the use of a plant-preferred translationinitiation context nucleotide sequence for the translation initiationcodon, and the insertion of a nucleotide sequence comprising a plantintron sequence upstream of the sequence encoding the polypeptide.

(2) Culturing a stably transformed duckweed plant or duckweed noduleculture that expresses at least one biologically active polypeptidecomprising a signal sequence that directs secretion of the polypeptideinto the culture medium, followed by recovering the biologically activepolypeptide from the culture medium. In one embodiment of this method,the nucleotide sequence encoding the biologically active polypeptide ismodified for enhanced expression in duckweed. Alternatively, in anotherembodiment the nucleotide sequence encoding the signal peptide ismodified for enhanced expression in duckweed. In one embodiment, thenucleotide sequence encoding both the biologically active polypeptideand the signal peptide are modified for enhanced expression in duckweed.

As another aspect, the present invention encompasses transformedduckweed plants or duckweed nodule cultures producing biologicallyactive polypeptides according to the methods above.

As another aspect, the present invention discloses methods and nucleicacid sequences for producing biologically active human α-2b-interferonin duckweed.

Definitions:

“Polypeptide” refers to any monomeric or multimeric protein or peptide.

“Biologically active polypeptide” refers to a polypeptide that has thecapability of performing one or more biological functions or a set ofactivities normally attributed to the polypeptide in a biologicalcontext. Those skilled in the art will appreciate that the term“biologically active” includes polypeptides in which the biologicalactivity is altered as compared with the native protein (e.g.,suppressed or enhanced), as long as the protein has sufficient activityto be of interest for use in industrial or chemical processes or as atherapeutic, vaccine, or diagnostics reagent. Biological activity can bedetermined by any method available in the art. For example, thebiological activity of members of the interferon family of proteins canbe determined by any of a number of methods including their interactionwith interferon-specific antibodies, their ability to increaseresistance to viral infection, or their ability to modulate thetranscription of interferon-regulated gene targets.

“Nucleotide sequence of interest” as used herein refers to anynucleotide sequence encoding a polypeptide intended for expression induckweed. For example, nucleotide sequences encoding therapeutic (e.g.,for veterinary or medical uses) or immunogenic (e.g., for vaccination)polypeptides can be expressed using transformed duckweed according tothe present invention.

The terms “expression” or “production” refer to the biosynthesis of agene product, including the transcription, translation, and assembly ofsaid gene product.

The term “duckweed” refers to members of the family Lemnaceae. Thisfamily currently is divided into four genera and 34 species of duckweedas follows: genus Lemna (L. aequinoctialis, L. disperma, L.ecuadoriensis, L. gibba, L. japonica, L. minor, L. miniscula, L.obscura, L. perpusilla, L. tenera, L. trisulca, L. turionifera, L.valdiviana); genus Spirodela (S. intermedia, S. polyrrhiza, S.punctata); genus Wolffia (Wa. angusta, Wa. arrhiza, Wa. australina, Wa.borealis, Wa. brasiliensis, Wa. columbiana, Wa. elongata, Wa. globosa,Wa. microscopica, Wa. neglecta) and genus Wolfiella (Wl. caudata, Wl.denticulata, Wl. gladiata, Wl. hyalina, Wl. lingulata, Wl. repunda, Wl.rotunda, and Wl. neotropica). Any other genera or species of Lemnaceae,if they exist, are also aspects of the present invention. Lemna speciescan be classified using the taxonomic scheme described by Landolt (1986)Biosystematic Investigation on the Family of Duckweeds: The family ofLemnaceae—A Monograph Study Geobatanischen Institut ETH, Stiftung Rubel,Zurich.

The term “duckweed nodule culture” as used herein refers to a culturecomprising duckweed cells wherein at least about 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, or 100% of the cells are differentiatedcells. A “differentiated cell,” as used herein, is a cell with at leastone phenotypic characteristic (e.g., a distinctive cell morphology orthe expression of a marker nucleic acid or protein) that distinguishesit from undifferentiated cells or from cells found in other tissuetypes. The differentiated cells of the duckweed nodule culture describedherein form a tiled smooth surface of interconnected cells fused attheir adjacent cell walls, with nodules that have begun to organize intofrond primordium scattered throughout the tissue. The surface of thetissue of the nodule culture has epidermal cells connect to each othervia plasmadesmata.

“Duckweed-preferred codons” as used herein refers to codons that have afrequency of codon usage in duckweed of greater than 17%.

“Lemna-preferred codons” as used herein refers to codons that have afrequency of codon usage in the genus Lemna of greater than 17%.

“Lemna gibba-preferred codons” as used herein refers to codons that havea frequency of codon usage in Lemna gibba of greater than 17% where thefrequency of codon usage in Lemna gibba was obtained from the CodonUsage Database (GenBank Release 113,0; athttp://www.kazusa.orjp/codon/cgibin/showcodon.cgi?species=Lemna+gibba+[gbpln]).

“Translation initiation codon” refers to the codon that initiates thetranslation of the mRNA transcribed from the nucleotide sequence ofinterest.

“Translation initiation context nucleotide sequence” as used hereinrefers to the identity of the three nucleotides directly 5′ of thetranslation initiation codon.

“Secretion” as used herein refers to translocation of a polypeptideacross both the plasma membrane and the cell wall of a host plant cell.

“Operably linked” as used herein in reference to nucleotide sequencesrefers to multiple nucleotide sequences that are placed in a functionalrelationship with each other. Generally, operably linked DNA sequencesare contiguous and, where necessary to join two protein coding regions,in reading frame.

A. Expression Cassettes

According to the present invention, stably transformed duckweed isobtained by transformation with a nucleotide sequence of interestcontained within an expression cassette. The expression cassettecomprises a transcriptional initiation region linked to the nucleic acidor gene of interest. Such an expression cassette is provided with aplurality of restriction sites for insertion of the gene or genes ofinterest (e.g., one gene of interest, two genes of interest, etc.) to beunder the transcriptional regulation of the regulatory regions. Theexpression cassette may encode a single gene of interest. In particularembodiments of the invention, the nucleic acid to be transferredcontains two or more expression cassettes, each of which encodes atleast one gene of interest.

The transcriptional initiation region, (e.g., a promoter) may be nativeor homologous or foreign or heterologous to the host, or could be thenatural sequence or a synthetic sequence. By foreign, it is intendedthat the transcriptional initiation region is not found in the wild-typehost into which the transcriptional initiation region is introduced. Asused herein a chimeric gene comprises a coding sequence operably linkedto a transcription initiation region that is heterologous to the codingsequence.

Any suitable promoter known in the art can be employed according to thepresent invention (including bacterial, yeast, fungal, insect,mammalian, and plant promoters). For example, plant promoters, includingduckweed promoters, may be used. Exemplary promoters include, but arenot limited to, the Cauliflower Mosaic Virus 35S promoter, the opinesynthetase promoters (e.g., nos, mas, ocs, etc.), the ubiquitinpromoter, the actin promoter, the ribulose bisphosphate (RubP)carboxylase small subunit promoter, and the alcohol dehydrogenasepromoter. The duckweed RubP carboxylase small subunit promoter is knownin the art (Silverthorne et al. (1990) Plant Mol. Biol. 15:49). Otherpromoters from viruses that infect plants, preferably duckweed, are alsosuitable including, but not limited to, promoters isolated from Dasheenmosaic virus, Chlorella virus (e.g., the Chlorella virus adeninemethyltransferase promoter; Mitra et al. (1994) Plant Mol. Biol. 26:85),tomato spotted wilt virus, tobacco rattle virus, tobacco necrosis virus,tobacco ring spot virus, tomato ring spot virus, cucumber mosaic virus,peanut stump virus, alfalfa mosaic virus, sugarcane baciliformbadnavirus and the like.

Finally, promoters can be chosen to give a desired level of regulation.For example, in some instances, it may be advantageous to use a promoterthat confers constitutive expression (e.g, the mannopine synthasepromoter from Agrobacterium tumefaciens). Alternatively, in othersituations, it may be advantageous to use promoters that are activatedin response to specific environmental stimuli (e.g., heat shock genepromoters, drought-inducible gene promoters, pathogen-inducible genepromoters, wound-inducible gene promoters, and light/dark-inducible genepromoters) or plant growth regulators (e.g., promoters from genesinduced by abscissic acid, auxins, cytokinins, and gibberellic acid). Asa further alternative, promoters can be chosen that give tissue-specificexpression (e.g., root, leaf, and floral-specific promoters).

The overall strength of a given promoter can be influenced by thecombination and spatial organization of cis-acting nucleotide sequencessuch as upstream activating sequences. For example, activatingnucleotide sequences derived from the Agrobacterium tumefaciens octopinesynthase gene can enhance transcription from the Agrobacteriumtumefaciens mannopine synthase promoter (see U.S. Pat. No. 5,955,646 toGelvin et al.). In the present invention, the expression cassette cancontain activating nucleotide sequences inserted upstream of thepromoter sequence to enhance the expression of the nucleotide sequenceof interest. In one embodiment, the expression cassette includes threeupstream activating sequences derived from the Agrobacterium tumefaciensoctopine synthase gene operably linked to a promoter derived from anAgrobacterium tumefaciens mannopine synthase gene (see U.S. Pat. No.5,955,646, herein incorporated by reference).

The transcriptional cassette includes in the 5′-3′ direction oftranscription, a transcriptional and translational initiation region, anucleotide sequence of interest, and a transcriptional and translationaltermination region functional in plants. Any suitable terminationsequence known in the art may be used in accordance with the presentinvention. The termination region may be native with the transcriptionalinitiation region, may be native with the nucleotide sequence ofinterest, or may be derived from another source. Convenient terminationregions are available from the Ti-plasmid of A. tumefaciens, such as theoctopine synthetase and nopaline synthetase termination regions. Seealso Guerineau et al. (1991) Mol. Gen. Genet. 262:141; Proudfoot (1991)Cell 64:671; Sanfacon et al. (1991) Genes Dev. 5:141; Mogen et al.(1990) Plant Cell 2:1261; Munroe et al. (1990) Gene 91:151; Ballas etal. (1989) Nucleic Acids Res. 17:7891; and Joshi et al. (1987) NucleicAcids Res. 15:9627. Additional exemplary termination sequences are thepea RubP carboxylase small subunit termination sequence and theCauliflower Mosaic Virus 35S termination sequence. Other suitabletermination sequences will be apparent to those skilled in the art.

Alternatively, the gene(s) of interest can be provided on any othersuitable expression cassette known in the art.

The expression cassettes may contain more than one gene or nucleic acidsequence to be transferred and expressed in the transformed plant. Thus,each nucleic acid sequence will be operably linked to 5′ and 3′regulatory sequences. Alternatively, multiple expression cassettes maybe provided.

Generally, the expression cassette will comprise a selectable markergene for the selection of transformed cells or tissues. Selectablemarker genes include genes encoding antibiotic resistance, such as thoseencoding neomycin phosphotransferase II (NEO) and hygromycinphosphotransferase (HPT), as well as genes conferring resistance toherbicidal compounds. Herbicide resistance genes generally code for amodified target protein insensitive to the herbicide or for an enzymethat degrades or detoxifies the herbicide in the plant before it canact. See DeBlock et al. (1987) EMBO J. 6:2513; DeBlock et al.(1989)Plant Physiol. 91:691; Fromm et al. (1990) BioTechnology 8:833;Gordon-Kamm et al. (1990) Plant Cell 2:603. For example, resistance toglyphosphate or sulfonylurea herbicides has been obtained using genescoding for the mutant target enzymes, 5-enolpyruvylshikimate-3-phosphatesynthase (EPSPS) and acetolactate synthase (ALS). Resistance toglufosinate ammonium, boromoxynil, and 2,4-dichlorophenoxyacetate(2,4-D) have been obtained by using bacterial genes encodingphosphinothricin acetyltransferase, a nitrilase, or a2,4-dichlorophenoxyacetate monooxygenase, which detoxify the respectiveherbicides.

For purposes of the present invention, selectable marker genes include,but are not limited to, genes encoding neomycin phosphotransferase II(Fraley et al. (1986) CRC Critical Reviews in Plant Science 4:1);cyanamide hydratase (Maier-Greiner et al. (1991) Proc. Natl. Acad. Sci.USA 88:4250); aspartate kinase; dihydrodipicolinate synthase (Perl etal. (1993) BioTechnology 11:715); bar gene (Toki et al. (1992) PlantPhysiol. 100:1503; Meagher et al. (1996) Crop Sci. 36:1367); tryptophandecarboxylase (Goddijn et al. (1993) Plant Mol. Biol. 22:907); neomycinphosphotransferase (NEO; Southern et al. (1982) J. Mol. Appl. Gen.1:327); hygromycin phosphotransferase (HPT or HYG; Shimizu et al. (1986)Mol. Cell. Biol. 6:1074); dihydrofolate reductase (DHFR; Kwok et al.(1986) Proc. Natl. Acad. Sci. USA 83:4552); phosphinothricinacetyltransferase (DeBlock et al. (1987) EMBO J. 6:2513);2,2-dichloropropionic acid dehalogenase (Buchanan-Wollatron et al.(1989) J. Cell. Biochem. 13D:330); acetohydroxyacid synthase (U.S. Pat.No. 4,761,373 to Anderson et al.; Haughn et al. (1988) Mol. Gen. Genet.221:266); 5-enolpyruvyl-shikimate-phosphate synthase (aroA; Comai et al.(1985) Nature 317:741); haloarylnitrilase (WO 87/04181 to Stalker etal.); acetyl-coenzyme A carboxylase (Parker et al. (1990) Plant Physiol.92:1220); dihydropteroate synthase (sulI; Guerineau et al. (1990) PlantMol. Biol. 15:127); and 32 kDa photosystem II polypeptide (psbA;Hirschberg et al. (1983) Science 222:1346 (1983).

Also included are genes encoding resistance to: gentamycin (e.g., aacC1,Wohlleben et al. (1989) Mol. Gen. Genet. 217:202-208); chloramphenicol(Herrera-Estrella et al. (1983) EMBO J. 2:987); methotrexate(Herrera-Estrella et al. (1983) Nature 303:209; Meijer et al. (1991)Plant Mol. Biol. 16:807); hygromycin (Waldron et al. (1985) Plant Mol.Biol. 5:103; Zhijian et al. (1995) Plant Science 108:219; Meijer et al.(1991) Plant Mol. Bio. 16:807); streptomycin (Jones et al. (1987) Mol.Gen. Genet. 210:86); spectinomycin (Bretagne-Sagnard et al. (1996)Transgenic Res. 5:131); bleomycin (Hille et al. (1986) Plant Mol. Biol.7:171); sulfonamide (Guerineau et al. (1990) Plant Mol. Bio. 15:127);bromoxynil (Stalker et al. (1988) Science 242:419); 2,4-D (Streber etal. (1989) BioTechnology 7:811); phosphinothricin (DeBlock et al. (1987)EMBO J. 6:2513); spectinomycin (Bretagne-Sagnard and Chupeau, TransgenicResearch 5:131).

The bar gene confers herbicide resistance to glufosinate-typeherbicides, such as phosphinothricin (PPT) or bialaphos, and the like.As noted above, other selectable markers that could be used in thevector constructs include, but are not limited to, the pat gene, alsofor bialaphos and phosphinothricin resistance, the ALS gene forimidazolinone resistance, the HPH or HYG gene for hygromycin resistance,the EPSP synthase gene for glyphosate resistance, the Hm1 gene forresistance to the Hc-toxin, and other selective agents used routinelyand known to one of ordinary skill in the art. See Yarranton (1992)Curr. Opin. Biotech. 3:506; Chistopherson et al. (1992) Proc. Natl.Acad. Sci. USA 89:6314; Yao et al. (1992) Cell 71:63; Reznikoff(1992)Mol. Microbiol. 6:2419; Barkley et al. (1980) The Operon 177-220; Hu etal. (1987) Cell 48:555; Brown et al. (1987) Cell 49:603; Figge et al.(1988) Cell 52:713; Deuschle et al. (1989) Proc. Natl. Acad. Sci. USA86:5400; Fuerst et al. (1989) Proc. Natl. Acad. Sci. USA 86:2549;Deuschle et al. (1990) Science 248:480; Labow et al. (1990) Mol. Cell.Biol. 10:3343; Zambretti et al. (1992) Proc. Natl. Acad. Sci. USA89:3952; Baim et al. (1991) Proc. Natl. Acad. Sci. USA 88:5072; Wyborskiet al. (1991) Nuc. Acids Res. 19:4647; Hillenand-Wissman (1989) Topicsin Mol. And Struc. Biol. 10:143; Degenkolb et al. (1991) Antimicrob.Agents Chemother. 35:1591; Kleinschnidt et al. (1988) Biochemistry27:1094; Gatz et al. (1992) Plant J. 2:397; Gossen et al. (1992) Proc.Natl. Acad. Sci. USA 89:5547; Oliva et al. (1992) Antimicrob. AgentsChemother. 36:913; Hlavka et al. (1985) Handbook of ExperimentalPharmacology 78; and Gill et al. (1988) Nature 334:721. Such disclosuresare herein incorporated by reference.

The above list of selectable marker genes are not meant to be limiting.Any selectable marker gene can be used in the present invention.

B. Modification of Nucleotide Sequences for Enhanced Expression inDuckweed

The present invention provides for the modification of the expressednucleotide sequence to enhance its expression in duckweed. One suchmodification is the synthesis of the nucleotide sequence of interestusing duckweed-preferred codons. Methods are available in the art forsynthesizing nucleotide sequences with plant-preferred codons. See,e.g., U.S. Pat. Nos. 5,380,831 and 5,436,391; Perlak et al. (1991) Proc.Natl. Acad. Sci. USA 15:3324; Iannacome et al. (1997) Plant Mol. Biol.34:485; and Murray et al., (1989) Nucleic Acids. Res. 17:477, hereinincorporated by reference. The preferred codons may be determined fromthe codons of highest frequency in the proteins expressed in duckweed.For example, the frequency of codon usage for Lemna gibba is found onthe web page:http://www.kazusa.orjp/codon/cgi-bin/showcodon.cgi?species=Lemna+gibba+[gbpln],and the frequency of codon usage for Lemna minor is found on the webpagehttp://www.kazusa.orjp/codon/cgibin/showcodon.cgi?species=Lemna+minor+[gbpln]and in Table 1. It is recognized that genes that have been optimized forexpression in duckweed and other monocots can be used in the methods ofthe invention. See, e.g., EP 0 359 472, EP 0 385 962, WO 91/16432;Perlak et al. (1991) Proc. Natl. Acad. Sci. USA 88:3324; lannacome etal. (1997) Plant Mol. Biol. 34:485; and Murray et al. (1989) Nuc. AcidsRes. 17:477, and the like, herein incorporated by reference. It isfurther recognized that all or any part of the gene sequence may beoptimized or synthetic. In other words, fully optimized or partiallyoptimized sequences may also be used. For example, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100% of the codons may be duckweed-preferred codons.In one embodiment, between 90 and 96% of the codons areduckweed-preferred codons. The coding sequence of the nucleotidesequence of interest may comprise codons used with a frequency of atleast 17% in Lemna gibba. In one embodiment, the modified nucleotidesequence is the human α-2B-interferon encoding nucleotide sequence shownin SEQ ID NO:2, which contains 93% duckweed preferred codons. TABLE 1Lemna gibba-preferred codons from GenBank Release 113 UUU 2.2 (4) UCU0.5 (1) UAU 2.2 (4) UGU 0.0 (0) UUC 50.5 (92) UCC 31.9 (58) UAC 40.1(73) UGC 17.6 (32) UUA 0.0 (0) UCA 0.5 (1) UAA 3.8 (7) UGA 1.6 (3) UUG2.7 (5) UCG 15.4 (28) UAG 0.0 (0) UGG 24.2 (44) CUU 0.5 (1) CCU 6.6 (12)CAU 0.5 (1) CGU 1.1 (2) CUC 39.0 (71) CCC 43.4 (79) CAC 6.6 (12) CGC26.9 (49) CUA 1.1 (2) CCA 2.2 (4) CAA 4.4 (8) CGA 1.1 (2) CUG 22.5 (41)CCG 20.9 (38) CAG 26.9 (49) CGG 7.7 (14) AUU 0.0 (0) ACU 3.3 (6) AAU 1.1(2) AGU 0.0 (0) AUC 33.5 (61) ACC 26.4 (48) AAC 37.9 (69) AGC 22.0 (40)AUA 0.0 (0) ACA 0.5 (1) AAA 0.0 (0) AGA 4.9 (9) AUG 33.5 (61) ACG 9.3(17) AAG 57.1 (104) AGG 6.0 (11) GUU 9.3 (17) GCU 7.1 (13) GAU 1.6 (3)GGU 1.1 (2) GUC 28.0 (51) GCC 73.6 (134) GAC 38.4 (70) GGC 46.7 (85) GUA0.0 (0) GCA 5.5 (10) GAA 2.2 (4) GGA 1.1 (2) GUG 34.0 (62) GCG 20.9 (38)GAG 62.6 (114) GGG 27.5 (50)

Other modifications can also be made to the nucleotide sequence ofinterest to enhance its expression in duckweed. These modificationsinclude, but are not limited to, elimination of sequences encodingspurious polyadenylation signals, exon-intron splice site signals,transposon-like repeats, and other such well characterized sequenceswhich may be deleterious to gene expression. The G-C content of thesequence may be adjusted to levels average for a given cellular host, ascalculated by reference to known genes expressed in the host cell. Whenpossible, the sequence may be modified to avoid predicted hairpinsecondary MRNA structures.

There are known differences between the optimal translation initiationcontext nucleotide sequences for translation initiation codons inanimals and plants and the composition of these translation initiationcontext nucleotide sequence can influence the efficiency of translationinitiation. See, for example, Lukaszewicz et al. (2000) Plant Science154:89-98; and Joshi et al. (1997); Plant Mol. Biol. 35:993-1001. In thepresent invention, the translation initiation context nucleotidesequence for the translation initiation codon of the nucleotide sequenceof interest may be modified to enhance expression in duckweed. In oneembodiment, the nucleotide sequence is modified such that the threenucleotides directly upstream of the translation initiation codon of thenucleotide sequence of interest are “ACC.” In a second embodiment, thesenucleotides are “ACA.”

Expression of a transgene in duckweed can also be enhanced by the use of5′ leader sequences. Such leader sequences can act to enhancetranslation. Translation leaders are known in the art and include, butare not limited to, picomavirus leaders, e.g., EMCV leader(Encephalomyocarditis 5′ noncoding region; Elroy-Stein et al. (1989)Proc. Natl. Acad. Sci USA 86:6126); potyvirus leaders, e.g., TEV leader(Tobacco Etch Virus; Allison et al. (1986) Virology 154:9); humanimmunoglobulin heavy-chain binding protein (BiP; Macajak and Samow(1991) Nature 353:90); untranslated leader from the coat protein mRNA ofalfalfa mosaic virus (AMV RNA 4; Jobling and Gehrke (1987) Nature325:622); tobacco mosaic virus leader (TMV; Gallie (1989) MolecularBiology of RNA, 23:56); potato etch virus leader (Tomashevskaya et al.(1993) J. Gen. Virol. 74:2717-2724); Fed-1 5′ untranslated region(Dickey (1992) EMBO J. 11:2311-2317); RbcS 5′ untranslated region(Silverthome et al. (1990) J. Plant. Mol. Biol. 15:49-58); and maizechlorotic mottle virus leader (MCMV; Lommel et al. (1991) Virology81:382). See also, Della-Cioppa et al. (1987) Plant Physiology 84:965.Leader sequence comprising plant intron sequence, including intronsequence from the maize dehydrogenase 1 gene, the castor bean catalasegene, or the Arabidopsis tryptophan pathway gene PAT1 has also beenshown to increase translational efficient in plants (Callis et al.(1987) Genes Dev. 1:1183-1200; Mascarenhas etal. (1990) Plant Mol. Biol.15:913-920). In one embodiment of the present invention, nucleotidesequence corresponding to nucleotides 1222-1775 of the maize alcoholdehydrogenase 1 gene (GenBank Accession Number X04049), set forth in SEQID NO: 1, is inserted upstream of the nucleotide sequence of interest toenhance the efficiency of its translation. In another embodiment, theexpression vector contains the leader from the Lemna gibbaribulose-bis-phosphate carboxylase small subunit 5B gene (Buzby et al.(1990) Plant Cell 2:805-814).

It is recognized that any of the duckweed expression-enhancingnucleotide sequence modifications described above can be used in thepresent invention, including any single modification or any possiblecombination of modifications. The phrase “modified for enhancedexpression in duckweed” as used herein refers to a nucleotide sequencethat contains any one or any combination of these modifications.

C. Signal Peptides

Secreted proteins are usually translated from precursor polypeptidesthat include a “signal peptide” that interacts with a receptor proteinon the membrane of the endoplasmic reticulum (ER) to direct thetranslocation of the growing polypeptide chain across the membrane andinto the endoplasmic reticulum for secretion from the cell. This signalpeptide is often cleaved from the precursor polypeptide to produce a“mature” polypeptide lacking the signal peptide. In an embodiment of thepresent invention, a biologically active polypeptide is expressed induckweed from a nucleotide sequence that is operably linked with anucleotide sequence encoding a signal peptide that directs secretion ofthe polypeptide into the culture medium. Plant signal peptides thattarget protein translocation to the endoplasmic reticulum (for secretionoutside of the cell) are known in the art. See, for example, U.S. Pat.No. 6,020,169 to Lee et al. In the present invention, any plant signalpeptide can be used to in target polypeptide expression to the ER. Insome embodiments, the signal peptide is the Arabidopsis thaliana basicendochitinase signal peptide (SEQ ID NO:8; amino acids 14-34 of NCBIProtein Accession No. BAA82823), the extension signal peptide (Stiefelet al. (1990) Plant Cell 2:785-793), the rice α-amylase signal peptide(SEQ ID NO:6; amino acids 1-31 of NCBI Protein Accession No. AAA33885),or a modified rice α-amylase signal sequence (SEQ ID NO:7). In anotherembodiment, the signal peptide corresponds to the signal peptide of asecreted duckweed protein.

Alternatively, a mammalian signal peptide can be used to targetrecombinant polypeptides expressed in genetically engineered duckweedfor secretion. It has been demonstrated that plant cells recognizemammalian signal peptides that target the endoplasmic reticulum, andthat these signal peptides can direct the secretion of polypeptides notonly through the plasma membrane but also through the plant cell wall.See U.S. Pat. Nos. 5,202,422 and 5,639,947 to Hiatt et al. In oneembodiment of the present invention, the mammalian signal peptide thattargets polypeptide secretion is the human α-2b-interferon signalpeptide (amino acids 1-23 of NCBI Protein Accession No. AAB59402 and SEQID NO:4).

In one embodiment, the nucleotide sequence encoding the signal peptideis modified for enhanced expression in duckweed, utilizing anymodification or combination of modifications disclosed in section Babove for the nucleotide sequence of interest. In another embodiment,the signal peptide the rice α-amylase encoded by the modified nucleotidesequence set forth in SEQ ID NO:3, which contains approximately 93%duckweed preferred codons.

The secreted biologically active polypeptide can be harvested from theculture medium by any conventional means known in the art and purifiedby chromatography, electrophoresis, dialysis, solvent-solventextraction, and the like.

D. Transformed Duckweed Plants and Duckweed Nodule Cultures

The stably transformed duckweed utilized in this invention can beobtained by any method known in the art. In one embodiment, the stablytransformed duckweed is obtained by one of the gene transfer methodsdisclosed in U.S. Pat. No. 6,040,498 to Stomp et al., hereinincorporated by reference. These methods include gene transfer byballistic bombardment with microprojectiles coated with a nucleic acidcomprising the nucleotide sequence of interest, gene transfer byelectroporation, and gene transfer mediated by Agrobacterium comprisinga vector comprising the nucleotide sequence of interest. In oneembodiment, the stably transformed duckweed is obtained via any one ofthe Agrobacterium-mediated methods disclosed in U.S. Pat. No. 6,040,498to Stomp et al. The Agrobacterium used is Agrobacterium tumefaciens orAgrobacterium rhizogenes.

It is preferred that the stably transformed duckweed plants utilized inthese methods exhibit normal morphology and are fertile by sexualreproduction. Preferably, transformed plants of the present inventioncontain a single copy of the transferred nucleic acid, and thetransferred nucleic acid has no notable rearrangements therein. Alsopreferred are duckweed plants in which the transferred nucleic acid ispresent in low copy numbers (i.e., no more than five copies,alternately, no more than three copies, as a further alternative, fewerthan three copies of the nucleic acid per transformed cell).

The stably transformed duckweed expresses a biologically active proteinhormone, growth factor, or cytokine, insulin, or growth hormone (inparticular, human growth hormone). Alternatively, the duckweed expressesbiologically active β-glucuronidase. The duckweed may expressbiologically active α-2b-interferon, for example human α-2b-interferonprecursor (NCBI Protein Accession No. AAB59402; set forth in SEQ IDNO:4) or mature human α-2b-interferon (amino acids 24-188 of NCBIProtein Accession No. AAB9402; set forth in SEQ ID NO:5) or biologicallyactive variants thereof.

By “biologically active variant” of human α-2b-interferon is intended apolypeptide derived from the native polypeptide by deletion (so-calledtruncation) or addition of one or more amino acids to the N-terminaland/or C-terminal end of the native protein; deletion or addition of oneor more amino acids at one or more sites in the native protein; orsubstitution of one or more amino acids at one or more sites in thenative protein. Biologically active variant α-2b-interferon polypeptidesencompassed by the present invention are biologically active, that isthey continue to possess the desired biological activity of the nativeα-2b-interferon including the ability to increase resistance to viralinfection, or the ability to modulate the transcription ofα-2b-interferon-regulated gene targets. Such biologically activevariants may result from, for example, genetic polymorphism or fromhuman manipulation. Biologically active variants of a nativeα-2b-interferon protein of the invention will have at least about 50%,60%, 65%, 70%, generally at least about 75%, 80%, 85%, preferably atleast about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, and more preferablyat least about 98%, 99% or more sequence identity to the amino acidsequence shown in SEQ ID NO:4 or SEQ ID NO:5. Thus, a biologicallyactive variant of an α-2b-interferon of the invention may differ fromthat protein by as few as 1-15 amino acid residues, as few as 1-10, suchas 6-10, as few as 5, as few as 4, 3, 2, or even 1 amino acid residue.Examples of biologically active variants of human α-interferon are knownin the art. See, for example, European patent EP211148B1, and U.S. Pat.Nos. 4,748,233, 4,801,685, 4,816,566, 4,973,479, 4,975,276, 5,089,400,5,098,703, 5,231,176, and 5,869,293; herein incorporated by reference.

The comparison of sequences and determination of percent identity andpercent similarity between two sequences can be accomplished using amathematical algorithm. In a preferred embodiment, the percent identitybetween two amino acid sequences is determined using the Needleman andWunsch (1970) J. Mol. Biol. 48:444-453 algorithm, which is incorporatedinto the GAP program in the GCG software package (available atwww.accelrys.com), using either a BLOSSUM62 matrix or a PAM250 matrix,and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1,2, 3, 4, 5, or 6. In yet another preferred embodiment, the percentidentity between two nucleotide sequences is determined using the GAPprogram in the GCG software package, using a BLOSUM62 scoring matrix(see Henikoff et al. (1989) Proc. Natl. Acad. Sci. USA 89:10915) and agap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4,5, or 6. A particularly preferred set of parameters (and the one thatshould be used if the practitioner is uncertain about what parametersshould be applied to determine if a molecule is within a sequenceidentity limitation of the invention) is using a BLOSUM62 scoring matrixwith a gap weight of 60 and a length weight of 3).

The percent identity between two amino acid or nucleotide sequences canalso be determined using the algorithm of E. Meyers and W. Miller (1989)CABIOS 4:11-17 which has been incorporated into the ALIGN program(version 2.0), using a PAM120 weight residue table, a gap length penaltyof 12 and a gap penalty of 4.

In one embodiment, the stably transformed duckweed plants or duckweednodule cultures express biologically active polypeptides that cannoteffectively be commercially produced by existing gene expressionsystems, because of cost or logistical constraints, or both. Forexample, some proteins cannot be expressed in mammalian systems becausethe protein interferes with cell viability, cell proliferation, cellulardifferentiation, or protein assembly in mammalian cells. Such proteinsinclude, but are not limited to, retinoblastoma protein, p53,angiostatin, and leptin. The present invention can be advantageouslyemployed to produce mammalian regulatory proteins; it is unlikely giventhe large evolutionary distance between higher plants and mammals thatthese proteins will interfere with regulatory processes in duckweed.Transgenic duckweed can also be used to produce large quantities ofproteins such as serum albumin (in particular, human serum albumin),hemoglobin, and collagen, which challenge the production capabilities ofexisting expression systems.

Finally, higher plant systems can be engineered to produce biologicallyactive multimeric proteins (e.g., monoclonal antibodies, hemoglobin,P450 oxidase, and collagen, and the like) far more easily than canmammalian systems. One exemplary approach for producing biologicallyactive multimeric proteins in duckweed uses an expression vectorcontaining the genes encoding all of the polypeptide subunits. See,e.g., During et al. (1990) Plant Mol. Biol. 15:281 and van Engelen etal. (1994) Plant Mol. Biol. 26:1701. This vector is then introduced intoduckweed cells using any known transformation method, such as aballistic bombardment or Agrobacterium-mediated transformation. Thismethod results in clonal cell lines that express all of the polypeptidesnecessary to assemble the multimeric protein. Accordingly, in someembodiments, the transformed duckweed contains one or more expressionvectors encoding the heavy and light chain of a monoclonal antibody orFab′ antibody fragment, and the monoclonal antibody or antibody fragmentis assembled in the duckweed plant from the expressed heavy and lightchain.

A variation on this approach is to make single gene constructs, mix DNAfrom these constructs together, then deliver this mixture of DNAs intoplant cells using ballistic bombardment or Agrobacterium-mediatedtransformation. As a further variation, some or all of the vectors mayencode more than one subunit of the multimeric protein (i.e., so thatthere are fewer duckweed clones to be crossed than the number ofsubunits in the multimeric protein). In an alternative embodiment, eachduckweed clone expresses at least one of the subunits of the multimericprotein, and duckweed clones secreting each subunit are culturedtogether and the multimeric protein is assembled in the media from thevarious secreted subunits. In some instances, it may be desirable toproduce less than all of the subunits of a multimeric protein, or even asingle protein subunit, in a transformed duckweed plant or duckweednodule culture, e.g., for industrial or chemical processes or fordiagnostic, therapeutic, or vaccination purposes.

EXPERIMENTAL

The following examples are offered for purposes of illustration, not byway of limitation.

Expression Vectors

The expression vector pBMSP- 1 used in some of the examples is describedin U.S. Pat. No. 5,955,646, herein incorporated by reference. ThepBMSP-1 transcriptional cassette contains three copies of atranscriptional activating nucleotide sequence derived from theAgrobacterium tumefaciens octopine synthase and, an additionaltranscriptional activating nucleotide sequence derived from theAgrobacterium tumefaciens mannopine synthase gene, a promoter regionderived from the Agrobacterium tumefaciens mannopine synthase gene, apolylinker site for insertion of the nucleotide sequence encoding thepolypeptide of interest, and a termination sequence derived from theAgrobacterium tumefaciens nopaline synthase gene (see, van Engelen etal. (1995) 4:288-290; Ni et al. (1995) Plant J. 7:661-76; and Luehrsenet al. (1991) Mol. Gen. Genet. 225:81-93, each of which is hereinincorporated by reference). The pBMSP-1 expression vector also containsa nucleotide sequence coding for neomycin phosphotransferase II as aselectable marker. Transcription of the selectable marker sequence isdriven by a promoter derived from the Agrobacterium tumefaciens nopalinesynthase gene.

The expression vector pBMSP-3, also used in some of the followingexamples, contains the components of the pBMSP-1 expression vectordescribed above and additionally contains a nucleotide sequencecorresponding to nucleotides 1222-1775 of the maize alcoholdehydrogenase gene (GenBank Accession Number X04049) inserted betweenthe promoter and the polylinker. This sequence is shown in SEQ ID NO: 1.

Expression Constructs for the Production of Human α-2b-Interferon inDuckweed

Table 2 shows the expression constructs used for the production of humanα-interferon in duckweed. TABLE 2 Construct Expression Name VectorSignal Peptide Interferon-encoding Sequence IFN01 pBMSP-1 NoneNon-optimized interferon (SEQ ID NO: 9; nucleotides 580-1077 of GenBankAccession No. J00207) IFN02 pBMSP-3 Non-optimized interferonNon-optimized interferon (SEQ ID NO: 10; nucleotides (SEQ ID NO: 9;nucleotides 580-1077 511-579 of GenBank Accession of GenBank AccessionNo. No. J00207) J00207) IFN03 pBMSP-3 Arabidopsis thaliana Non-optimizedinterferon endochitinase (SEQ ID NO: 11; (SEQ ID NO: 9; nucleotides580-1077 nucleotides 338-399 of GenBank of GenBank Accession No.Accession number AB023460 J00207) with an additional “A” added to the 3′end of the sequence) IFN05 pBMSP-3 Modified rice α-amylase*Non-optimized interferon (encoding the amino acid (SEQ ID NO: 9;nucleotides 580-1077 sequence shown in SEQ ID of GenBank Accession No.NO: 7) J00207) IFN07 pBMSP-3 Wild type rice α-amylase Non-optimizedinterferon (nucleotides 34-126 of GenBank (SEQ ID NO: 9; nucleotides580-1077 Accession No. M24286, of GenBank Accession No. encoding theamino acid J00207) sequence shown in SEQ ID NO: 6) IFN08 pBMSP-3Optimized wild type rice Non-optimized interferon α-amylase (SEQ ID NO:3) (SEQ ID NO: 9; nucleotides 580-1077 of GenBank Accession No. J00207)IFN09 pBMSP-3 Optimized wild type rice Optimized interferon α-amylase(SEQ ID NO: 3) (SEQ ID NO: 2) IFN10 pBMSP-3 None Optimized interferon(SEQ ID NO: 2) IFN11 pBMSP-1 Optimized wild type rice Optimizedinterferon α-amylase (SEQ ID NO: 3) (SEQ ID NO: 2) IFN12 pBMSP-1 NoneOptimized interferon (SEQ ID NO: 2) IFN053 modified Optimized wild typerice Optimized interferon pBMSP-3** α-amylase (SEQ ID NO: 3) (SEQ ID NO:2)*The nucleotide sequence encoding the modified rice α-amylase signalpeptide corresponds to nucleotides 34-126 of NCBI Accession No. M24286,except that nucleotides 97-102 have been changed from “CTTGGC” to“ATCGTC.”**For construct IFN053, the 5′-mas leader in pBMPSP3 was replaced withthe leader from the ribulose-bis-phosphate carboxylase small subunit 5Bgene of Lemna gibba (nucleotides 689-751 of NCBI Accession No. S45167,Buzby et al. (1990) Plant Cell 2: 805-814; SEQ ID NO: 16).Transformation of Duckweed

Duckweed fronds or duckweed nodule cultures (derived from Lemna minorstrain 8627 in these examples) are transformed with the expressionconstructs described above using Agrobacteria-mediated transformationmethods. Agrobacterium tumefaciens strain C58Z707, a disarmed, broadhost range C58 strain (Hepburn et al. (1985) J. Gen. Microbiol.131:2961-2969) is used for transformation in these examples. Theexpression constructs described above are mobilized into A. tumefaciensby electroporation, or by a triparental mating procedure using E. coliMM294 harboring the mobilizing plasmid pRK2013 (Hoekema et al. (1983)Nature 303: 179-180; Ditta et al. (1980) Proc. Natl. Acad. Sci. USA 77:7347-7350). C58Z707 strains comprising the expression constructsdescribed above are streaked on AB minimal medium (Chilton et al.,(1974) Proc. Nat. Acad. Sci. USA 71: 3672 -3676) or in YEB medium (1 g/Lyeast extract, 5 g/L beef extract, 5 g/L peptone, 5 g/L sucrose, 0.5 g/LMgSO₄) containing streptomycin at 500 mg/L, spectinomycin at 50 mg/L andkanamycin sulfate at 50 mg/L and grown overnight at28° C.

In these examples, Lemna minor strain 8627 is used for transformationalthough any Lemna strain can be used. Fronds are grown on liquid Schenkand Hildebrandt medium (Schenk and Hildebrandt (1972) Can. J Bot.50:199) containing 1% sucrose and 10 μM indoleacetic acid at 23° C.under a 16-hour light/8-hour dark photoperiod with light intensity ofapproximately 40 μM/m².sec. For inoculation, individual fronds areseparated from clumps and floated in inoculation media for approximately2 to 20 minutes. The inoculating medium is Schenk and Hildebrandt medium(pH 5.6) supplemented with 0.6 M mannitol and 100 μM acetosyringone,with the appropriate Agrobacterium tumefaciens strain comprising theexpression construct present at a concentration of about 1×10⁹ cells/ml.These fronds are then transferred to Schenk and Hildebrandt medium (pH5.6) containing 1% sucrose, 0.9% agar, and 20 mg/L acetosyringone andare co-cultivated for 3 or 4 days in the dark at 23° C.

Following co-cultivation, the fronds are transferred for recovery toSchenk and Hildebrandt medium or Murashige and Skoog medium (Murashigeand Skoog (1962) Physiol. Plant. 15:473) supplemented with 200 μg/mlkanamycin sulfate. Fronds are decontaminated from infecting Agrobacteriaby transferring the infected tissue to fresh medium with antibioticevery 2-4 days. The fronds are incubated on this medium forapproximately four weeks under conditions of low light (1-5 μM/m².sec).

Duckweed nodule cultures for transformation are produced as follows.Duckweed fronds are separated, the roots are cut off with a sterilescalpel, and the fronds are placed, ventral side down, on Murashige andSkoog medium (catalog number M-5519; Sigma Chemical Corporation, St.Louis, Mo.) pH 5.6, supplemented with 5 μM 2,4-dichlorophenoxyaceticacid, 0.5 μM 1-Phenyl-3(1,2,3-thiadiazol-5-yl) urea thidiazuron (SigmaP6186), 3% sucrose, 0.4 Difco Bacto-agar (Fisher Scientific), and 0.15%Gelrite (Sigma). Fronds are grown for 5-6 weeks. At this time, thenodules (small, yellowish cell masses) appear, generally from thecentral part of the ventral side. This nodule tissue is detached fromthe mother frond and cultured in Murashige and Skoog medium supplementedwith 3% sucrose, 0.4% Difco Bacto-agar, 0.15% Gelrite, 1 μM2,4-dichlorophenoxyacetic acid, and 2 μM benzyladenine.

Duckweed nodule cultures are transformed as follows. The appropriateAgrobacterium tumefaciens strain is grown on potato dextrose agar or YEBagar with 50 mg/L kanamycin and 100 μM acetosyringone, and resuspendedin Murashige and Skoog medium supplemented with 0.6 M Mannitol and 100μM acetosyringone. Nodule culture tissue is inoculated by immersing inthe solution of resuspended bacteria for 1-2 minutes, blotted to removeexcess fluid, and plated on co-cultivation medium consisting ofMurashige and Skoog medium supplemented with auxin and cytokininoptimized to promote nodule growth and 100 μM acetosyringone. See,Yamamoto et al. (2001) In Vitro Cell Dev. Biol. Plant 37:349-353.

For selection, nodule culture tissue is transferred to regenerationmedium Murashige and Skoog medium with 3% sucrose, 1 μM2,4-dichlorophenoxyacetate, 2 μM benzyladenine, 0.4% Difco Bacto-Agar,0.15% Gelrite 500 mg/L cefotaxime, and 200 mg/L kanamycin sulfate andcultured for approximately four weeks under continuous light (20-40μM/m²·sec). The nodule tissue is transferred every 7 days to freshculture medium. Selection is complete when the nodule tissue showsvigorous growth on the selection agent. In some examples, thetransformed duckweed nodule cultures are transferred directly toregeneration medium for selection, instead of undergoing selection inco-cultivation medium.

For regeneration of transformed duckweed, the selected nodule culture istransferred to regeneration medium (0.5× Schenk and Hildebrandt mediumsupplemented with 1% sucrose and 200 mgs/L kanamycin) to organize andproduce plants. The nodule culture is incubated on regeneration mediumunder full light for approximately 3 weeks, until fronds appear. Fullyorganized fronds are transferred to liquid Schenk and Hildebrandt mediumwith 1-3% sucrose and incubated under full light for further clonalproliferation.

Detection of Biologically-Active Interferon Produced from DuckweedFronds or Duckweed Nodule Culture

Biologically-active interferon is detected by various assays, includinga solid phase sandwich immunoassay as described in Staehlin et al.(1981) Methods Enzymol. 79:589-594 and Kelder et al. (1986) MethodsEnzymol. 119:582-587, herein incorporated by reference, and a cytopathiceffect inhibition assay (described in Tovey et al. (1978) Nature276:270-272, herein incorporated by reference. Secreted interferon iscollected from the duckweed culture medium, while non-secretedinterferon is collected from ground-up or lysed duckweed plants orduckweed nodule tissue.

A solid phase sandwich immunoassay for interferon was performed using akit from PBL Laboratories (New Brunswick, N.J.) according to themanufacturer's instructions. Briefly, interferon is captured by anantibody bound to the microtiter plate wells. A second antibody is thenused to reveal the bound antibody. An anti-secondary antibody conjugatedto horseradish peroxidase (HRP) is then used to mark the interferon.Tetramethyl benzidine (TMB) initiates a peroxidase-catalyzed colorchange so that the interferon level can be observed and compared with astandard. A monoclonal antibody specific for α-2b-interferon (Cat. No.11105, PBL Laboratories) is used for this assay in the present examples.

A cytopathic effect inhibition assay was performed according to themethods of Tovey et al. (1978) Nature 276:270-272. Briefly, serialtwo-fold dilutions of the preparation to be assayed are diluted in a 96well microtiter plate (Falcon Inc) in a volume of 100 μl of Eaglesminimal essential medium (Life Technologies Inc) supplemented with 2%fetal calf serum (Life Technologies Inc) in parallel with serial twofold dilutions of the US National Institutes of Health human IFN alphainternational reference preparation (G-002-901-527). Twenty thousandhuman amnion cells (line WISH) are then added to each well of themicrotiter plate in a volume of 100 μl of medium with 2% fetal calfserum. The cells are incubated over-night in an atmosphere of 5% CO₂ inair at 37° C., the medium is removed and replaced with 200 μl of mediumwith 2% fetal calf serum containing vesicular stomatitis virus at amultiplicity of infection of 0.1. The cells are further incubatedover-night in an atmosphere of 5% CO₂ in air at 37° C. and thecytopathic effect due to virus replication is then evaluated under alight microscope. Interferon titers are expressed as the reciprocal ofthe IFN dilution which gave 50% protection against the cytopathiceffects of the virus. Interferon titers are expressed in internationalreference units by reference to the titer of the reference preparation.

The following examples demonstrate the expression of biologically activeinterferon, human growth hormone, and antibodies in duckweed.

Example 1

A study was performed to determine culture IFN levels in media andtissue at various time points in a batch culture. A set of 20-30 ml 175oz.-culture jars were inoculated on Day 0 with 20 fronds of a linepreviously identified as expressing detectable levels of humanα-2b-interferon (IFN). The cultures were grown under autotrophic,buffered conditions with continuous high light provided byplant/aquarium fluorescent grow bulbs. At each time point—days 5, 7, 13,15, and 18—the fresh weight and media volume were measured for fourcultures. From each culture, media and tissue samples were obtained anda plant protease inhibitor cocktail was added. The tissue samples wereground and spun cold. The supernatant was collected. Media and tissueextracts were stored at −70° C. until all samples were collected. IFNlevels in media and tissue extracts were determined on the same dayusing the solid phase sandwich immunoassay described above. Totalculture IFN in media and tissue was calculated by multiplying measuredIFN concentrations and the volume of media and the fresh weight for theculture, respectively. FIG. 1 shows the relative IFN levels on days 7,13, 15, and 18 compared to day 5. The last time point represents theaverage value for three cultures instead of four due to loss of oneculture.

Example 2

A study was performed to determine culture IFN levels in media andtissue at various time points in a batch culture. A set of 24-30 ml 175oz.-culture jars were inoculated on Day 0 with 20 fronds of the sameline as in Example 1. The cultures were grown under autotrophic,unbuffered conditions with continuous high light provided byplant/aquarium fluorescent grow bulbs. At each time point—days 7, 10,12, 14, 17, and 19—the fresh weight and media volume were measured forfour cultures. From each culture, media and tissue samples were obtainedand a plant protease inhibitor cocktail was added. The tissue sampleswere ground and spun cold. The supernatant was collected. Media andtissue extracts were stored at −70° C. until all samples were collected.IFN levels in media and tissue extracts were determined on the same dayusing the solid phase sandwich immunoassay described above. Totalculture IFN in media and tissue was calculated by multiplying measuredIFN concentrations and the volume of media and the fresh weight for theculture, respectively. FIG. 2 shows the relative IFN levels on days 14,17, and 19 compared to day 12. Media IFN levels on day 7 and day 10 werebelow the range of the extended range protocol for the immunoassay.

Example 3

Duckweed lines transformed with the expression constructs listed inTable 2 were produced using the methods described above. Thesetransformed duckweed lines were grown for 14 days under autotrophicconditions. Bovine serum albumin at a concentration of 0.2 mg/ml wasincluded in the growth media. On day 14, media and tissue extracts wereprepared as described in Example 1, and the interferon levels in theseextracts were determined using the solid phase sandwich immunoassay asdescribed above. Table 3 gives the number of clonal duckweed linesassayed and the mean media interferon concentration for each expressionconstruct. Table 4 shows the interferon levels within the duckweedtissue for the duckweed lines transformed with the interferon expressionconstructs that did not contain a signal peptide (IFN01, IFN10, andIFN12). Both the mean interferon level for all clonal lines assayed forthe designated construct, and the interferon level for thetop-expressing line are shown. TABLE 3 Mean Interferon Expression # ofClonal Lines Concentration Construct Tested (ng/ml) IFN01 41 0 IFN02 752.3 IFN03 41 0.18 IFN05 44 2.5 IFN07 41 1.5 IFN08 41 1.4 IFN09 41 30.3IFN10 41 0 IFN11 39 9.9 IFN12 41 0

TABLE 4 Mean Value Top Expresser % of total % of total IFN Constructsoluble protein soluble protein IFN01 0.00003 0.0001 IFN10 0.000640.0074 IFN12 0.00014 0.001

The biological activity of the interferon produced by these transformedduckweed lines was assayed by the cytopathic effect inhibition assaydescribed above. Table 5 gives the results for the top expressing linefor each construct. The interferon activity is shown for the media forthose constructs containing a signal peptide and the tissue for thoseconstructs lacking a signal peptide. TABLE 5 Top Expresser Source IU/ml(media) IFN Construct material IU/mg total protein (tissue) IFN01 Tissue40 IFN02 Media 16,000 IFN03 Media 320 IFN05 Media 6,400 IFN07 Media6,000 IFN08 Media 3,200 IFN09 Media 200,000 IFN10 Tissue 19,300 IFN11Media 30,000 IFN12 Tissue 150

The following features can be noted for the data in this example: (1)Secretion of interferon was detected only for those duckweed linestransformed with expression constructs containing a signal peptide.Compare, for example, the media interferon concentration for IFN02,IFN03, IFN05, IFN07, IFN08, IFN 09 and IFN011 with that for IFN01,IFN10, and IFN12. (2) Secretion of interferon was detected for allexpression constructs containing a signal peptide, with the native humaninterferon signal sequence producing the highest level of secretedinterferon. Compare, for example, the interferon levels produced byIFN02 with those produced by IFN03 and IFN05. (3) The use ofduckweed-preferred codons leads to enhanced interferon expression.Compare, for example, the interferon levels produced by IFN09 with thosefor IFN05. (4) Higher expression levels were achieved by the codonoptimization of more than just the signal peptide. Compare, for example,IFN08 with IFN09. (5) Including the maize alcohol dehydrogenase intronsequence in the expression construct resulted in higher levels ofinterferon expression. Compare, for example, IFN09 with IFN11 and IFN10with IFN12. (6) No statistically significant difference was observedbetween the interferon levels expressed from the construct containingthe modified α-amylase signal peptide (IFN05) in comparison with thatcontaining the wild-type alpha amylase signal peptide (IFN07).

Example 4

A study was performed to determine the levels of IFN expressed fromtransgenic duckweed grown at bioproduction scales. Transgenic duckweedplants were generated using IFN expression constructs IFN02, IFN05,IFN09, IFN10, and IFN53 (see Table 2). A minimum of 40 independenttransgenic lines was screened for each of these constructs. The linesproducing the highest levels of IFN expression were analyzed further.The concentration of IFN in the media and tissue was determined by ELISAas described elsewhere herein.

Table 6 summarizes the IFN expression on both research and bioproductionscales for the constructs described above. Because Lemna is unique inthat it grows in a very dilute inorganic media with a low proteincontent, the expression values in this Table are defined by thepre-purification titer. In the case of IN53, IFN represents over 30% ofthe total media proteins. TABLE 6 Expression of IFN in Lemna MeanAverage Concentration for 2 Media IFN Week Screening Trialsconcentration for Top (determined by Expressing Line (mg/L)^(c)ELISA)^(b) Research Research Bio- Tissue Expression Scale Scaleproduction Media (mg/kg construct (2 weeks) (3 weeks) Scale (mg/L)^(c)tissue)^(a) IFN02 2.0 — — 0.12 23.3 IFN05 1.1 — — 0.13 86.7 IFN09 24.8 60  30 1.51 164 IFN10 <0.1 — — <0.0001 99.3 IFN53 100 300 500 15.3^(d)—^(a)Based on 1 g of tissue yielding 20 mg of protein.^(b)Based on recovery of 10 ml of media and 1 go of tissue per screeningtrial.^(c)Expressed as a pre-purification titer.^(d)1 week screening trial.

The antiviral activity of the duckweed-produced IFN was determined asfollows. HuH7 cells were incubated for 24 hours at 37° C. with 1,000IU/ml of unpurified IFN from the duckweed media or Intron® A (Schering)as a control. The IFN was then removed and the cells were washed twice.The cells were subsequently infected with an RNA virus selected fromEncephalomyocarditis virus (EMCV), vesicular stomatitis virus (VSV), orSindbis at a multiplicity of infection of 1.0 for 1 hour, at which timethe virus innoculum was removed. The cells were then washed three timesand allowed to grow for 24 hours at 37° C. The cells were harvested,lysed by six freeze/thaw cycles, and then cell debris was removed bycentrifugation. Serial dilutions of the virus were then assayed fortheir cytopathic effect on monkey CV1 Vero cells. The duckweed-producedIFN exhibited similar antiviral activity to that observed for Intron®A.

The antiproliferative activity of the duckweed-produced IFN wasdetermined as follows. Interferon-sensitive Daudi cells were seeded inmicrotiter plates at an initial concentration of 50,000 cells/ml. Theculture were then either left untreated or were treated with 1000 IU ofunpurified duckweed-produced IFN, Intron® A, or an equivalent volume ofcontrol media derived from non-transgenic plants grown under the sameconditions as for the transgenic plants. After four days, the number ofviable and dead cells were determined by the trypan blue-exclusionviability test.

Example 5

Human growth hormone (hGH) was expressed in duckweed as follows. Anexpression vector for hGH was constructed by replacing the 5′-mas leaderin pBMPSP3 with the leader from the ribulose-bis-phosphate carboxylasesmall subunit 5B gene of Lemna gibba as described above for the IFN053construct. The expression cassette contained synthetic duckweedcodon-optimized sequences encoding the hGH signal peptide (SEQ ID NO:12)and the hGH polypeptide (SEQ ID NO:14). The amino acid sequence of theencoded proteins are given in SEQ ID NO: 13 (hGH signal peptide) and SEQID NO: 15 (hGH mature polypeptide). Transgenic duckweed lines expressinghGH were generated as described above.

The hGH concentration in the duckweed media was determined by ELISAusing a kit available from Roche Diagnostics, Manheim, Germany. Thelevels of secreted hGH in the plant growth media were 609 mg/L beforepurification, and approximately 50% of the expressed hGH remained in theduckweed tissue. The duckweed-produced hGH protein products were alsoanalyzed by Western blot analysis using a mouse anti-human hGH antibodyavailable from United States Biological in Swampscott, Mass., and wereshown to co-migrate with recombinant hGH produced in E. coli.

Activity of the duckweed-expressed hGH was determined by an NB2 lymphomacell bioassay essentially as described by Aston et al. (1986) 64:227-34.The duckweed-produced hGH in an unpurified media sample showed asignificant level of biological activity, similar to that seen forpurified hGH produced in E. coli.

Example 6

A Fab′ fragment was expressed in Lemna from a dicistronic vectorcontaining codon-optimized heavy and light chains of the Fab′ fragment.The expression vector for the Fab′ was constructed by replacing the5′-mas leader in pBMPSP3 with the leader from the ribulose-bis-phosphatecarboxylase small subunit 5B gene of Lemna gibba as described above forthe IFN053 construct. The second expression cassette for expression ofthe light chain contained its own promoter and terminator and wasinserted into the modified pBMSP3 vector in a head to tail configurationwith the first expression cassette. A duckweed codon optimized sequenceencoding a rice α-amylase signal peptide (SEQ ID NO:3) was joined to thesequences encoding the heavy and light chain. Both the heavy and lightchain sequences also contained codons optimized for expression in Lemnaminor.

A quantitative western blot showed that the expressed Fab′ fragmentrepresented up to 4% of the soluble protein, which translates into 8.62grams of Fab′ fragment/kg of dry weight. The majority of the Fab′ wasretained in the duckweed tissue. The western blot analysis showed thatthe duckweed-produced Fab′ fragment co-migrated with the Fab′ fragmentproduced in E. coli. The antigen-binding activity of the Fab′ fragmentwas confirmed by using the duckweed-produced Fab′ fragment to detectantigen on a Western blot.

Example 7

A mAb was expressed in Lemna using the expression vector described abovefor Fab′ expression. The sequences encoding the heavy and light chainswere both codon-optimized with Lemna-preferred codons. A quantitativewestern blot showed that the expressed mAb fragment represented up to2.8% of the soluble protein, which translates into 5.60 grams of mAbfragment/kg of dry weight. The majority of the mAb was retained in theduckweed tissue. A crude preparation of Lemna-produced mAb was analyzedby Western blot analysis. The preparation contained a high proportion offully assembled mAb. In addition, the Lemna-produced mAb co-migratedwith the mAb produced in CHO cells. The antigen-binding activity of themAb was confirmed by using the duckweed-produced Fab′ fragment to detectantigen on a Western blot.

All publications and patent applications mentioned in the specificationare indicative of the level of those skilled in the art to which thisinvention pertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

1. A method of producing human growth hormone in a duckweed plant culture or a duckweed nodule culture, comprising the steps of: (a) culturing within a duckweed culture medium a duckweed plant culture or a duckweed nodule culture, wherein said duckweed plant culture or said duckweed nodule culture is stably transformed to express said human growth hormone, and wherein said human growth hormone is expressed from a nucleotide sequence comprising a coding sequence for the human growth hormone and an operably linked coding sequence for a signal peptide that directs secretion of the human growth hormone into the culture medium; and (b) collecting said human growth hormone from the duckweed culture medium.
 2. The method of claim 1, wherein said human growth hormone is secreted into the duckweed culture medium.
 3. The method of claim 1, wherein said nucleotide sequence has at least one attribute selected from the group consisting of: (a) duckweed-preferred codons in the coding sequence for said human growth hormone; (b) duckweed-preferred codons in the coding sequence for said signal peptide; (c) a translation initiation codon that is flanked by a plant-preferred translation initiation context nucleotide sequence; and (d) an operably linked nucleotide sequence comprising a plant intron that is inserted upstream of the coding sequence; and (e) an operably linked nucleotide sequence comprising the leader sequence from the ribulose-bis-phosphate carboxylase small subunit 5B gene of Lemna gibba.
 4. The method according to claim 3, wherein said duckweed-preferred codons are Lemna gibba-preferred codons or Lemna minor-preferred codons.
 5. The method according to claim 4, wherein at least one coding sequence selected from the coding sequence for said polypeptide and the coding sequence for said signal peptide comprises between 70-100% Lemna gibba-preferred codons or Lemna minor-preferred codons.
 6. The method according to claim 3, wherein said plant-preferred translation initiation context nucleotide sequence consists of the nucleotide sequence “ACC” or “ACA”, wherein said context is positioned immediately adjacent to of the 5′ end of the translation initiation codon.
 7. The method according to claim 3, wherein said operably linked nucleotide sequence comprising said plant intron is the sequence set forth in SEQ ID NO:1.
 8. A method of producing an antibody in a duckweed plant culture or a duckweed nodule culture, comprising the steps of: (a) culturing within a duckweed culture medium a duckweed plant culture or a duckweed nodule culture, wherein said duckweed plant culture or said duckweed nodule culture is stably transformed to express said antibody, and wherein said antibody is expressed from one or more nucleotide sequences comprising a coding sequence for a chain of the antibody and an operably linked coding sequence for a signal peptide that directs secretion of the antibody into the culture medium; and (b) collecting said antibody from the duckweed culture.
 9. The method of claim 8, wherein said one or more nucleotide sequences have at least one attribute selected from the group consisting of: (a) duckweed-preferred codons in the coding sequence for the chain of the antibody; (b) duckweed-preferred codons in the coding sequence for said signal peptide; (c) a translation initiation codon that is flanked by a plant-preferred translation initiation context nucleotide sequence; (d) an operably linked nucleotide sequence comprising a plant intron that is inserted upstream of the coding sequence; and (e) an operably linked nucleotide sequence comprising the leader sequence from the ribulose-bis-phosphate carboxylase small subunit 5B gene of Lemna gibba.
 10. The method according to claim 9, wherein said duckweed-preferred codons are Lemna gibba-preferred codons or Lemna minor-preferred codons.
 11. The method according to claim 10, wherein the coding sequence comprises between 70% and 100% Lemna gibba-preferred codons or Lemna minor-preferred codons.
 12. The method according to claim 9, wherein said plant-preferred translation initiation context nucleotide sequence consists of the nucleotide sequence “ACC” or “ACA”, wherein said context is positioned immediately adjacent to the 5′ end of the translation initiation codon.
 13. The method according to claim 9, wherein said operably linked nucleotide sequence comprising said plant intron is the sequence set forth in SEQ ID NO:1.
 14. The method according to claim 8, wherein said duckweed frond culture or duckweed nodule culture expresses and assembles the heavy chain and light chain of the antibody.
 15. The method according to claim 8, wherein said antibody is a Fab′ fragment.
 16. The method according to claim 8, wherein said antibody is a mAb
 17. The method according to claim 8, wherein the antibody is a human antibody.
 18. The method according to claim 1, wherein said human growth hormone has at least 90% sequence identity with the amino acid sequence set forth in SEQ ID NO:
 15. 19. The method according to claim 18, wherein the human growth hormone has the has the amino acid sequence set forth in SEQ ID NO:15.
 20. The method according to claim 1, wherein the coding sequence for the human growth hormone comprises the nucleotide sequence set forth in SEQ ID NO:14.
 21. The method according to claim 1, wherein said signal peptide sequence has the amino acid sequence set forth in SEQ ID NO:13.
 22. The method according to claim 21, wherein the coding sequence for the signal peptide comprises the nucleotide sequence set forth in SEQ ID NO:14.
 23. The method according to claim 8, wherein said signal peptide sequence has the sequence set forth in SEQ ID NO:6.
 24. The method according to claim 23, wherein the coding sequence for the signal peptide comprises the nucleotide sequence set forth in SEQ ID NO:3.
 25. The stably transformed duckweed plant culture or duckweed nodule culture according to claim
 1. 26. The stably transformed duckweed plant culture or duckweed nodule culture according to claim 25, wherein said duckweed plant culture or duckweed nodule culture is selected from the group consisting of the genus Spirodela, genus Wolffia, genus Wolfiella, and genus Lemna.
 27. The stably transformed duckweed plant culture or duckweed nodule culture according to claim 26, wherein said duckweed plant culture or duckweed nodule culture is selected from the group consisting of Lemna minor, Lemna miniscula, Lemna aequinoctialis, and Lemna gibba.
 28. The stably transformed duckweed plant culture or duckweed nodule culture according to claim
 8. 29. An isolated nucleic acid molecule comprising a nucleotide sequence encoding an amino acid sequence selected from the group consisting of: (a) the amino acid sequence set forth in SEQ ID NO:15; (b) the amino acid sequence of a variant of the human growth hormone sequence shown in SEQ ID NO:15, wherein said variant has at least about 90% sequence identity with the amino acid sequence set forth in SEQ ID NO:15; and wherein said nucleotide sequence comprises duckweed-preferred codons.
 30. A nucleic acid molecule comprising a nucleotide sequence encoding the human growth hormone signal peptide amino acid sequence set forth in SEQ ID NO:13; wherein said nucleotide sequence comprises duckweed-preferred codons.
 31. A method of producing human a-interferon in a duckweed plant culture or a duckweed nodule culture, comprising the steps of: (a) culturing within a duckweed culture medium a duckweed plant culture or a duckweed nodule culture, wherein said duckweed plant culture or said duckweed nodule culture is stably transformed to express said human α-interferon, and wherein said α-interferon is expressed from a nucleotide sequence comprising the leader sequence from the ribulose-bis-phosphate carboxylase small subunit 5B gene of Lemna gibba operably linked to a coding sequence for the human growth hormone, and an operably linked coding sequence for a signal peptide that directs secretion of the α-interferon into the culture medium; and (b) collecting said α-interferon from the duckweed culture medium.
 32. The method of claim 31, wherein said α-interferon is secreted into the duckweed culture medium.
 33. The method of claim 31, wherein said nucleotide sequence has at least one attribute selected from the group consisting of: (a) duckweed-preferred codons in the coding sequence for said α-interferon; (b) duckweed-preferred codons in the coding sequence for said signal peptide; (c) a translation initiation codon that is flanked by a plant-preferred translation initiation context nucleotide sequence; and (d) an operably linked nucleotide sequence comprising a plant intron that is inserted upstream of the coding sequence.
 34. The method according to claim 33, wherein said duckweed-preferred codons are Lemna gibba-preferred codons or Lemna minor-preferred codons.
 35. The method according to claim 34, wherein at least one coding sequence selected from the coding sequence for said polypeptide and the coding sequence for said signal peptide comprises between 70-100% Lemna gibba-preferred codons or Lemna minor-preferred codons.
 36. The method according to claim 33, wherein said plant-preferred translation initiation context nucleotide sequence consists of the nucleotide sequence “ACC” or “ACA”, wherein said context is positioned immediately adjacent to of the 5′ end of the translation initiation codon.
 37. The method according to claim 33, wherein said operably linked nucleotide sequence comprising said plant intron is the sequence set forth in SEQ ID NO:1.
 38. The method of claim 31, wherein the leader sequence from the ribulose-bis-phosphate carboxylase small subunit 5B gene of Lemna gibba is the nucleotide sequence shown in SEQ ID NO:16.
 39. The method of claim 31, wherein the signal peptide has the amino acid sequence set forth in SEQ ID NO:6.
 40. The stably transformed duckweed plant culture or duckweed nodule culture according to claim
 31. 41. Human growth hormone produced according to the method of claim
 1. 42. An antibody produced according to the method of claim 8
 43. α-interferon produced according to the method of claim
 31. 44. A method of enhancing the expression of a biologically active polypeptide in duckweed, said method comprising culturing a duckweed plant culture or a duckweed nodule culture, wherein said duckweed plant culture or said duckweed nodule culture is stably transformed to express said biologically active polypeptide and wherein said biologically active polypeptide is expressed from a nucleotide sequence comprising a coding sequence for the biologically active polypeptide and an operably linked nucleotide sequence comprising the leader from the ribulose-bis-phosphate carboxylase small subunit gene of Lemna gibba.
 45. The method of claim 44, wherein said leader from the ribulose-bis-phosphate carboxylase small subunit gene of Lemna gibba has the nucleotide sequence set forth in SEQ ID NO:16. 